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Dense masses of chromatin are present in the nuclei of many cells during interphase; dense chromatin is also present in the chromosomes of cells during mitosis. Relatively little RNA synthesis takes place in the dense chromatin of interphase cells,1' 2 and apparently none at all in the dense chromatin of metaphase chromosomes. It has been shown that chromatin threads of thymus nuclei are bound together into dense masses by lysine-rich histones.3 In this paper we present evidence that in metaphase chromosomes, too, chromatin threads are held together by lysine-rich histones.The experimental procedure, as in the previous work, was to remove selectively the lysine-rich histones (which comprise about 20% of the total histone) and then to examine the chromosomes with the electron microscope. Removal of lysinerich histones loosened the structure of the chromatin in metaphase chromosomes. The special role of lysine-rich histones in binding chromatin threads together was shown by restoring histones to histone-depleted chromosomes. Only the lysinerich histones caused dense chromatin to reappear, although both arginine-rich and lysine-rich histones combined with histone-depleted chromosomes.This paper is a sequel to one on chromatin in the interphase nucleus. Some of the procedures followed here are more fully described in the earlier paper.3 Materials and Methods.-Preparation of metaphase chromosomes: HeLa cells of the S-3 strain were carried in suspension culture according to standard procedures in Eagle's minimal essential medium supplemented with 5% calf serum, antibiotics, and 4 ml\I glutamine. The cell line utilized was obtained from Microbiological Associates, and the media from Microbiological Associates and Grand Island Biological Co. Cells were synchronized according to the excess thymidine procedure of Puck.4 Thymidine was added to exponentially growing cell cultures to 2 mM final concentration. After 24 hr the cells were centrifuged down and resuspended in normal medium. After 5 hr, cell division begins to occur and all the cells in the population divide within the next several hours. At 15 hr from the addition of normal medium, the cells were again exposed to excess thymidine for 24 hr. No cell division at all takes place in the presence of excess thymidine. After removal of thymidine and return to normal medium, a second wave of division, even more highly synchronized than the first, occurs in the cell population. For the experiments described, it was necessary to employ only one cycle of excess thymidine treatment, however.To obtain metaphase chromosomes, a synchronized cell population was treated with colcemide (0.06 ,g/ml) 7 hr after relief of the excess thymidine block. Control cultures were allowed to divide in the absence of colcemide, and in. all the experiments cited approximately 100% of the cells underwent division during the next 5-6 hr. Colcemidetreated cells progressed only to metaphase and accumulated at this stage. When harvested, the cell populations thus consisted of up to 90% metaphase cells...

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